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1.  Regular heartbeat rhythm at the heartbeat initiation stage is essential for normal cardiogenesis at low temperature 
Background
The development of blood flow in the heart is crucial for heart function and embryonic survival. Recent studies have revealed the importance of the extracellular matrix and the mechanical stress applied to the valve cushion that controls blood flow to the formation of the cardiac valve during embryogenesis. However, the events that trigger such valve formation and mechanical stress, and their temperature dependence have not been explained completely. Medaka (Oryzias latipes) inhabits a wide range of East Asia and adapts to a wide range of climates. We used medaka embryos from different genomic backgrounds and analyzed heartbeat characteristics including back-and-forth blood flow and bradyarrhythmia in embryos incubated at low temperature. We also used high-speed imaging analysis to examine the heartbeat of these animals after transient exposure to low temperature.
Results
Embryos of the Hd-rR medaka strain exhibited back-and-forth blood flow in the heart (blood regurgitation) after incubation at 15°C. This regurgitation was induced by exposure to low temperature around the heartbeat initiation period and was related to abnormalities in the maintenance or pattern of contraction of the atrium or the atrioventricular canal. The Odate strain from the northern Japanese group exhibited normal blood flow after incubation at 15°C. High-speed time-lapse analysis of the heartbeat revealed that bradyarrhythmia occurred only in Hd-rR embryos incubated at 15°C. The coefficient of contraction, defined as the quotient of the length of the atrium at systole divided by its length at diastole, was not affected in either strain. The average heart rate after removing the effect of arrhythmia did not differ significantly between the two strains, suggesting that the mechanical stress of individual myocardial contractions and the total mechanical stress could be equivalent, regardless of the presence of arrhythmia or the heart rate. Test-cross experiments suggested that this circulation phenotype was caused by a single major genomic locus.
Conclusions
These results suggest that cardiogenesis at low temperature requires a constant heartbeat. Abnormal contraction rhythms at the stage of heartbeat initiation may cause regurgitation at later stages. From the evolutionary viewpoint, strains that exhibit normal cardiogenesis during development at low temperature inhabit northern environments.
doi:10.1186/1471-213X-14-12
PMCID: PMC3936829  PMID: 24564206
Heartbeat; Medaka; Blood regurgitation; Cold adaptation; Cardiogenesis
2.  Allelic Expression Changes in Medaka (Oryzias latipes) Hybrids between Inbred Strains Derived from Genetically Distant Populations 
PLoS ONE  2012;7(5):e36875.
Variations in allele expressions between genetically distant populations are one of the most important factors which affects their morphological and physiological variations. These variations are caused by natural mutations accumulated in their habitats. It has been reported that allelic expression differences in the hybrids of genetically distant populations are different from parental strains. In that case, there is a possibility that allelic expression changes lead to novel phenotypes in hybrids. Based on genomic information of the genetically distant populations, quantification and comparison of allelic expression changes make importance of regulatory sequences (cis-acting factors) or upstream regulatory factors (trans-acting modulators) for these changes clearer. In this study, we focused on two Medaka inbred strains, Hd-rR and HNI, derived from genetically distant populations and their hybrids. They are highly polymorphic and we can utilize whole-genome information. To analyze allelic expression changes, we established a method to quantify and compare allele-specific expressions of 11 genes between the parental strains and their reciprocal hybrids. In intestines of reciprocal hybrids, allelic expression was either similar or different in comparison with the parental strains. Total expressions in Hd-rR and HNI were tissue-dependent in the case of HPRT1, with high up-regulation of Hd-rR allele expression in liver. The proportion of genes with differential allelic expression in Medaka hybrids seems to be the same as that in other animals, despite the high SNP rate in the genomes of the two inbred strains. It is suggested that each tissue of the strain difference in trans-acting modulators is more important than polymorphisms in cis-regulatory sequences in producing the allelic expression changes in reciprocal hybrids.
doi:10.1371/journal.pone.0036875
PMCID: PMC3349633  PMID: 22590630
3.  Dual control by a single gene of secondary sexual characters and mating preferences in medaka 
BMC Biology  2009;7:64.
Background
Animals utilize a wide variety of tactics to attract reproductive partners. Behavioral experiments often indicate an important role for visual cues in fish, but their molecular basis remains almost entirely unknown. Studies on model species (such as zebrafish and medaka) allow investigations into this fundamental question in behavioral and evolutionary biology.
Results
Through mate-choice experiences using several laboratory strains of various body colors, we successfully identified one medaka mutant (color interfere; ci) that is distinctly unattractive to reproductive partners. This unattractiveness seems to be due to reduced orange pigment cells (xanthophores) in the skin. The ci strain carries a mutation on the somatolactin alpha (SLa) gene, therefore we expected over-expression of SLa to make medaka hyper-attractive. Indeed, extremely strong mating preferences were detected in a choice between the ci and SLa-transgenic (Actb-SLa:GFP) medaka. Intriguingly, however, the strains showed opposite biases; that is, the mutant and transgenic medaka liked to mate with partners from their own strain, similar to becoming sexually isolated.
Conclusion
This study spotlighted SLa as a novel mate-choice gene in fish. In addition, these results are the first demonstration of a single gene that can pleiotropically and harmoniously change both secondary sexual characters and mating preferences. Although theoretical models have long suggested joint evolution of linked genes on a chromosome, a mutation on a gene-regulatory region (that is, switching on/off of a single gene) might be sufficient to trigger two 'runaway' processes in different directions to promote (sympatric) speciation.
doi:10.1186/1741-7007-7-64
PMCID: PMC2761876  PMID: 19788724
4.  Medaka: a promising model animal for comparative population genomics 
BMC Research Notes  2009;2:88.
Background
Within-species genome diversity has been best studied in humans. The international HapMap project has revealed a tremendous amount of single-nucleotide polymorphisms (SNPs) among humans, many of which show signals of positive selection during human evolution. In most of the cases, however, functional differences between the alleles remain experimentally unverified due to the inherent difficulty of human genetic studies. It would therefore be highly useful to have a vertebrate model with the following characteristics: (1) high within-species genetic diversity, (2) a variety of gene-manipulation protocols already developed, and (3) a completely sequenced genome. Medaka (Oryzias latipes) and its congeneric species, tiny fresh-water teleosts distributed broadly in East and Southeast Asia, meet these criteria.
Findings
Using Oryzias species from 27 local populations, we conducted a simple screening of nonsynonymous SNPs for 11 genes with apparent orthology between medaka and humans. We found medaka SNPs for which the same sites in human orthologs are known to be highly differentiated among the HapMap populations. Importantly, some of these SNPs show signals of positive selection.
Conclusion
These results indicate that medaka is a promising model system for comparative population genomics exploring the functional and adaptive significance of allelic differentiations.
doi:10.1186/1756-0500-2-88
PMCID: PMC2683866  PMID: 19426554
6.  WDR55 Is a Nucleolar Modulator of Ribosomal RNA Synthesis, Cell Cycle Progression, and Teleost Organ Development 
PLoS Genetics  2008;4(8):e1000171.
The thymus is a vertebrate-specific organ where T lymphocytes are generated. Genetic programs that lead to thymus development are incompletely understood. We previously screened ethylnitrosourea-induced medaka mutants for recessive defects in thymus development. Here we report that one of those mutants is caused by a missense mutation in a gene encoding the previously uncharacterized protein WDR55 carrying the tryptophan-aspartate-repeat motif. We find that WDR55 is a novel nucleolar protein involved in the production of ribosomal RNA (rRNA). Defects in WDR55 cause aberrant accumulation of rRNA intermediates and cell cycle arrest. A mutation in WDR55 in zebrafish also leads to analogous defects in thymus development, whereas WDR55-null mice are lethal before implantation. These results indicate that WDR55 is a nuclear modulator of rRNA synthesis, cell cycle progression, and embryonic organogenesis including teleost thymus development.
Author Summary
Medaka, Oryzias latipes, is a small freshwater fish that is useful for studies of forward and reverse genetics. The availability of various inbred strains is the distinct advantage of medaka over zebrafish, especially in studies of the immune system. The thymus is a primary immune organ that is unique to vertebrates and supports the generation of T lymphocytes. Defective thymus development tends to cause abnormal T lymphocyte development, resulting in immunodeficiency or autoimmunity. However, the molecular pathways underlying thymus development have not been fully uncovered. Here we report the positional cloning of a gene responsible for one of the thymus-defective medaka mutants. We find that the hkc phenotype is caused by a missense mutation in a gene encoding the previously uncharacterized protein WDR55. Our results indicate that WDR55 is a novel nucleolar modulator of rRNA biosynthesis, cell cycle progression, and vertebrate development of organs, including teleost thymus. These results not only provide evidence of the existence of a new mechanism of rRNA production but also demonstrate that the malfunction of WDR55 causes cell cycle arrest and developmental failure, including defective thymus development.
doi:10.1371/journal.pgen.1000171
PMCID: PMC2515640  PMID: 18769712
7.  Radiation Hybrid Maps of Medaka Chromosomes LG 12, 17, and 22 
Abstract
The Medaka is an excellent genetic system for studies of vertebrate development and disease and environmental and evolutionary biology studies. To facilitate the mapping of markers or the cloning of affected genes in Medaka mutants identified by forward-genetic screens, we have established a panel of whole-genome radiation hybrids (RHs) and RH maps for three Medaka chromosomes. RH mapping is useful, since markers to be mapped need not be polymorphic and one can establish the order of markers that are difficult to resolve by genetic mapping owing to low genetic recombination rates. RHs were generated by fusing the irradiated donor, OLF-136 Medaka cell line, with the host B78 mouse melanoma cells. Of 290 initial RH clones, we selected 93 on the basis of high retention of fragments of the Medaka genome to establish a panel that allows genotyping in the 96-well format. RH maps for linkage groups 12, 17, and 22 were generated using 159 markers. The average retention for the three chromosomes was 19% and the average break point frequency was ∼33 kb/cR. We estimate the potential resolution of the RH panel to be ∼186 kb, which is high enough for integrating RH data with bacterial artificial chromosome clones. Thus, this first RH panel will be a useful tool for mapping mutated genes in Medaka.
doi:10.1093/dnares/dsm012
PMCID: PMC2779899  PMID: 17591665
Medaka; radiation hybrid mapping; genetic mapping; BAC
8.  Generation of medaka gene knockout models by target-selected mutagenesis 
Genome Biology  2006;7(12):R116.
A reverse genetics approach for the routine generation of medaka (Oryzias latipes) gene knockouts is described and applied to create a cryopreserved resource containing knockouts for most medaka genes.
We have established a reverse genetics approach for the routine generation of medaka (Oryzias latipes) gene knockouts. A cryopreserved library of N-ethyl-N-nitrosourea (ENU) mutagenized fish was screened by high-throughput resequencing for induced point mutations. Nonsense and splice site mutations were retrieved for the Blm, Sirt1, Parkin and p53 genes and functional characterization of p53 mutants indicated a complete knockout of p53 function. The current cryopreserved resource is expected to contain knockouts for most medaka genes.
doi:10.1186/gb-2006-7-12-r116
PMCID: PMC1794429  PMID: 17156454
9.  Determination of Pyrimidine Dimers in Escherichia coli and Cryptosporidium parvum during UV Light Inactivation, Photoreactivation, and Dark Repair 
Applied and Environmental Microbiology  2001;67(10):4630-4637.
UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.
doi:10.1128/AEM.67.10.4630-4637.2001
PMCID: PMC93213  PMID: 11571166
10.  Neurocytotoxic effects of iron-ions on the developing brain measured in vivo using medaka (Oryzias latipes), a vertebrate model 
Purpose: Exposure to heavy-ion radiation is considered a critical health risk on long-term space missions. The developing central nervous system (CNS) is a highly radiosensitive tissue; however, the biological effects of heavy-ion radiation, which are greater than those of low-linear energy transfer (LET) radiation, are not well studied, especially in vivo in intact organisms. Here, we examined the effects of iron-ions on the developing CNS using vertebrate organism, fish embryos of medaka (Oryzias latipes).
Materials and methods: Medaka embryos at developmental stage 28 were irradiated with iron-ions at various doses of 0-1.5 Gy. At 24 h after irradiation, radiation-induced apoptosis was examined using an acridine orange (AO) assay and histo-logically. To estimate the relative biological effectiveness (RBE), we quantified only characteristic AO-stained rosette-shaped apoptosis in the developing optic tectum (OT). At the time of hatching, morphological abnormalities in the irradiated brain were examined histologically.
Results: The dose-response curve utilizing an apoptotic index for the iron-ion irradiated embryos was much steeper than that for X-ray irradiated embryos, with RBE values of 3.7-4.2. Histological examinations of irradiated medaka brain at 24 h after irradiation showed AO-positive rosette-shaped clusters as aggregates of condensed nuclei, exhibiting a circular hole, mainly in the marginal area of the OT and in the retina. However, all of the irradiated embryos hatched normally without apparent histological abnormalities in their brains.
Conclusion: Our present study indicates that the medaka embryo is a useful model for evaluating neurocytotoxic effects on the developing CNS induced by exposure to heavy iron-ions relevant to the aerospace radiation environment.
doi:10.3109/09553002.2011.584944
PMCID: PMC3169016  PMID: 21770703
High-LET radiation; apoptosis; embryonic brain; medaka; relative biological effectiveness (RBE)

Results 1-10 (10)